Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNF alpha and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNF alpha and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naive and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNF alpha and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3(+) tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45(+) tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naive and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNF alpha and mIL-2 compared to monotherapies. This observation was verified by striking CD3(+) TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNF alpha and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNF alpha and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.

Recent approaches in the treatment of cancer focus on involving the immune system to control the tumor growth. The administration of immunotherapies, like checkpoint inhibitors, has shown impressive results in the long term survival of patients. Cancer vaccines are being investigated as further tools to prime tumor-specific immunity. Biomaterials show potential as adjuvants in the formulation of vaccines, and biomimetic elements derived from the membrane of tumor cells may widen the range of antigens contained in the vaccine. Here, we show how mice presenting an aggressive melanoma tumor model treated twice with the complete nanovaccine formulation showed control on the tumor progression, while in a less aggressive model, the animals showed remission and control on the tumor progression, with a modification in the immunological profile of the tumor microenvironment. We also prove that co-administration of the nanovaccine together with a checkpoint inhibitor increases the efficacy of the treatment (87.5% of the animals responding, with 2 remissions) compared to the checkpoint inhibitor alone in the B16.OVA model. Our platform thereby shows potential applications as a cancer nanovaccine in combination with the standard clinical care treatment for melanoma cancers.

Chimeric antigen receptor (CAR) T cells are genetically modified usually autologous T cells expressing de novo designed CAR that binds a specific antigen on the surface of the cancer cells, inducing T cell receptor-independent activation and cytotoxic response against the targeted cancer cells. While CAR T cells have been shown to offer effective treatment in acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma, several resistance mechanisms can lead to CAR T cell exhaustion characterized by impaired functions and the expression of inhibitory receptors. The Finnish Red Cross Blood Service has developed novel CARs, differing in structure from the ones currently published. Since the evasion of CAR T cell exhaustion is considered one of the key objectives in the development of CAR T cell therapy, this Master’s thesis project aimed to create a working method to determine the exhaustion of CAR T cells in vitro after long-term repeated stimulation. In order to induce and measure exhaustion, CAR T cells were produced and activated ex vivo in the presence of IL-2 or IL-7/IL-15 cytokines, cultured long-term and repeatedly stimulated by exposure to target cells. CAR T cell cytotoxicity and expansion were determined and the expression of various inhibitory receptors was analyzed. The method enabled the comparison of the designed CAR T cell candidates and the positive control CD19-CD28ζ CAR T cells in long-term cytotoxic potency. In addition, it helped to reveal the surprising difference between IL-2 and IL-7/IL-15 cytokines and their impact on CAR T cell exhaustion. Although CAR T cells produced with IL-2 had poorer expansion during CAR T cell production than CAR T cells produced with IL-7/IL-15, they showed lower expression of exhaustion-related markers supported by better survival, proliferation and cytotoxic activity during long-term repeated stimulation assay.

The human immune system can provide a powerful tool in developing therapies against various cancers. Even though the idea of an immune system actively searching for and disposing of potential mutated tumor cells is over a century old, only recent developments in various fields such as mass spectrometry, immuno-checkpoint blockade strategies and in silico modelling have enabled the realization of the full potential of recruiting immune system to fight cancer and the possibilities of personalized therapies. These therapeutic methods, including but not limited to oncolytic virus therapies, T-cell therapies and cancer vaccines, are based on the body’s ability to recognize mutated antigen peptides presented on the cell surface by MCH-receptors (also known as HLA-receptors in humans) and the disposal of the malignant cells by cytotoxic T-cells. Thus, the capability to map the individual HLA-presented peptidome and differentiate the immunogenic peptides is a foundation for this plethora of therapies and is in focus of ongoing research. This master thesis is a part of a project aiming to set up immunoaffinity-purification/MS based method in order to analyse the ligandome and determine T-cell recognized cancer associated antigens from tumor cells. Objectives of the work: 1. Characterizing tumor cell lines. 2. Immunological assay set up. 3. Collecting cell culture material for the ligandome affinity purification. 4. In silico prediction if the immunogenicity of selected peptides and assessing their source proteins. Methods used: 1. Cell culture. 2. FACS-analysis. 3. MTS-viability assay. 4. Immunological assays (ELISA, ELISPOT). 5. Immunological bioinformatics analysis tools (IEDB) and database search (UniPROT). Results: 1. Flow cytometric analysis provided essential information of the cell line HLA-1 expression. Additional information of PD-L1 expression can be used to evaluate cell line’s immune-evasion abilities. Preliminary MTS assay is used to determine linear range and optimal time frame for the PBMC/cancer cell co-culture killing assay. 2. Interferon γ cytokine secretion was determined by ELISPOT to assess PBMC response against known antigens in a preliminary experiment to approximate usable range for the following antigen specific PBMC assays. ELISA is used to confirm the presence of HLA-I receptors in the ligandome affinity purification eluates and to estimate the efficacy of purification. 3. Feasibility of in silico methods in the prediction of immunogenic peptides was explored. The experiments provided information that can be applied to the further development of the immune ligandome discovery project. In silico methods were successfully used to characterize previously identified HLA-restricted peptides and one previously identified immunogenic T-cell epitope. Even if the data acquired in silico can be considered only nominally verified at this stage, the results are encouraging.

Cancer immunotherapy refers to therapy strategies that utilise the mechanisms of the immune system to treat cancer patients. The benefits of the approach include the possibility for specific targeting and utilisation of the host immune system. The treatment methods include cancer vaccines, oncolytic viruses (OVs), cell-based immunotherapies and antibodies. The interplay between the cancer and the immune system has been observed crucial for the progress of the cancer and the success of immunotherapies. An immune inflamed tumour microenvironment has been observed beneficial for the success of several therapy methods. Many immunotherapy methods rely on detecting tumour specific antigens that are used to guide the therapy agent to the target site. This strategy poses challenges when considering tumour immune evasion mechanisms, which can cause downregulation of target antigens, and heterogeneity of tumour cells and patients. OVs have the advantage of not requiring predetermined target structures to exert their effect to the tumour cells. They cause direct tumour cell lysis and induce immune responses, and may be modified to express additional genes, including immunostimulatory agents. However, virus-related immunosuppressive mechanisms and a rapid viral clearance may limit their effects. A Western Reserve (WR) Vaccinia virus (VACV) is a highly oncolytic virus strain but the virus has been observed to suppress the function of the cyclic guanosine monophosphate adenosine monophosphate synthase – stimulator of interferon genes (cGAS STING) innate immune pathway which has been shown to have a significant role in anti-tumour immune responses. The aim of this study was to create a WR VACV encoding a dominantly active (D A) STING and to determine whether the virus is capable of activating the cGAS STING pathway. The effects were compared to a corresponding virus vvdd tdTomato that does not have the STING encoding gene. The pathogenicity of viruses was controlled by a double deletion of the thymidine kinase and vaccinia growth factor genes which restricts the virus replication to tumour cells. Transgene fragments were cloned from template plasmids by polymerase chain reactions (PCRs) and joined together in a Gibson Assembly (GA) reaction to form a STING-P2A-eGFP gene insert. The insert was attached to a shuttle vector pSC65-tdTomato by restriction enzyme digestion, ligation and transformation in Escherichia coli. The correct transgene plasmid construct was verified by Sanger sequencing and PCRs. The transgene was inserted to a modified WR VACV vvdd-tdTomato-hDAI by a homologous recombination. The newly created VVdd STING-P2A-eGFP virus was purified by plaque purification. The STING protein expression was studied by an immunocytochemistry (ICC) assay. The immune signalling pathway activation was examined by testing nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation in RAW-Blue cells and dendritic cell activation and maturation in JAWS II cells. The cell viability after iinfection was studied with four cell lines; A549, B16-F10, HEK293 and MB49. The D-A STING expressing virus was produced successfully. The ICC experiment verified the capability of the VVdd STING-P2A eGFP to produce the STING protein in the infected cells. The preliminary findings indicate that the VVdd STING-P2A-eGFP virus activates the NF-κB signalling in the RAW-Blue cells and that the activation is dependent on the STING expression. The activation level is relative to the infection concentration at MOI range 0,001 to 0,1. The findings suggest that the VVdd-STING-eGFP virus can induce innate immune signalling via the STING pathway. The reference virus did not activate the signalling. The in vitro experiments also indicated that the STING virus may induce DC activation and maturation. We observed a trend of CD86 and CD40 expression upregulation on the JAWS II DCs. The effects to the cell viability were inconclusive. More studies should be conducted to verify the results. The effects of the virus should be studied in more advanced cancer models that take into account the complexity of the immune system. These preliminary results indicate the that the VVdd-STING-P2A-eGFP virus could stimulate the immune signalling through the STING pathway.

Syöpätautien yleisyys lisääntyy kaikkialla maailmassa väestön vanhetessa ja Suomessakin todetaan vuosittain noin 35 000 uutta syöpää, joihin kuolee noin 13 000 henkeä. Syövän lääkehoidon painopiste on siirtymässä laajalti elimistön soluja tuhoavista sädehoidosta ja solunsalpaajista kohti potilaan omaa immuunipuolustusta syöpää vastaan ohjaavia immuno-onkologisia lääkkeitä. Immuno-onkologisilla lääkkeillä, kuten tarkastuspiste-estäjiin kuuluvilla PD-1-vasta-aineilla on saavutettu jopa pysyviä remissioita useassa vaikeahoitoisessa syövässä. Valitettavasti vain pieni osa immunologisilla syöpälääkkeillä hoidetuista potilaista saa niiden täyden hyödyn ja hoitovasteen laajentaminen koko potilasjoukkoon on osoittautunut ongelmalliseksi. Tutkielmani kirjallisuuskatsauksessa käsitellään rintasyöpää yleisesti, sekä syvennytään rintasyövän prekliinisiin tutkimusmalleihin ja rintasyöpäpotilaan immuno-onkologiavasteen ennustetekijöihin. Kokeellisen laboratoriotutkimusosuuden tavoitteina oli korreloida rintasyöpänäytteiden leukosyytti-infiltraation taso kasvaimen Myc- ja PD-L1-ekspressioon, mitata solulinjojen ja tutkimusryhmämme kehittämien PDEC-eksplanttien immuuniaktivaatiota, sekä arvioida näiden tutkimusten tulosten avulla rintasyöpäpotilaan kasvainnäytteestä kasvattamiemme PDEC-kudosviljelmien potentiaalia rintasyövän immuno-onkologisessa tutkimuksessa. Löysin tutkimalla primäärikasvainten ja PDEC-eksplanttien ominaisuuksia immunohistokemialla sekä qRT-PCR:llä korrelaation primäärikasvaimen leukosyyttimäärän sekä PDEC-viljelmien qRT-PCR:llä mitatun immuuniaktiivisuuden välille: kasvaimen korkea leukosyyttipitoisuus vaikuttaa olevan yhteydessä PDEC-viljelmien suurempaan immuuniaktiivisuuteen. Mielenkiintoista oli havaita, että vaikka immuunisolujen aktivoimiseen käytetyn Immunocultin tulisi aktivoida kaikkia T-soluja, niin edes runsas kasvaimen strooman leukosyytti-infiltraatio ei aiheuttanut qRT-PCR:llä tehdyissä immuuniaktivaatiomittauksissa menetelmän käyttöä häiritseviä vääriä positiivisia tuloksia. Tulosteni perusteella PDEC-eksplantit kykenevät kuvaamaan rintasyöpäpotilaiden kasvainkohtaisia immuuniaktivaatioeroja, mikä korostaa niiden soveltuvuutta immuno-onkologiseen tutkimukseen.

BackgroundImmune checkpoint inhibitors (ICIs), primarily anti-PD-1, are currently used to treat patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients benefit from these costly therapies. Therefore, there is an unmet need to better understand the effect of ICIs on immune effector cells. This study aimed to investigate the effect of a PD-1 antibody and an IDO1 inhibitor on different lymphocyte populations (NK, CD4(+), and CD8(+) T cells) in term of migration, cytotoxicity, and cytokine release in the presence of HNSCC cells. MethodsUsing a microfluidic chip, we injected HSC-3 cells (an oral tongue squamous cell carcinoma cell line) embedded in a human tumor-derived matrix "myogel/fibrin" together with NK, CD4(+), and CD8(+) T cells in separate channels. The two channels were connected with microchannels. The PD-1 antibody nivolumab and IDO1 inhibitor epacadostat were added to the microfluidic chips. Lymphocyte migration and cytotoxicity were examined under fluorescent microscopy and cytokine release was measured using a FirePlex Human Discovery Cytokines Immunoassay. ResultsEpacadostat significantly increased the migration and infiltration of NK and CD4(+) T cells, but not CD8(+) T cells, towards the cancer cells. Nivolumab did not exhibit a similar effect. While CD8(+) T cells alone showed near to no migration, adding CD4(+) T cells enhanced migration towards the cancer cells. There was a mild nonsignificant increase in apoptosis of HSC-3 cells after adding epacadostat to lymphocytes. In contrast, HSC-3 proliferation was not affected by lymphocytes regardless of ICIs. Nivolumab significantly increased release of MIP1-alpha, IL-6, and IL-8 from NK, CD4(+), and CD8(+) T cells, respectively. ConclusionsThis study revealed that each subpopulation of lymphocytes respond differently to ICIs. We also revealed the subpopulation of lymphocytes responsible for the increases in specific serum cytokines after ICI treatment.

Cytokines have proven to be effective for cancer therapy, however whilst low-dose monotherapy with cytokines provides limited therapeutic benefit, high-dose treatment can lead to a number of adverse events. Interleukin 7 has shown promising results in clinical trials, but anti-cancer effect was limited, in part due to a low concentration of the cytokine within the tumor. We hypothesized that arming an oncolytic adenovirus with Interleukin 7, enabling high expression localized to the tumor microenvironment, would overcome systemic delivery issues and improve therapeutic efficacy. We evaluated the effects of Ad5/3-E2F-d24-hIL7 (TILT-517) on tumor growth, immune cell activation and cytokine profiles in the tumor microenvironment using three clinically relevant animal models and ex vivo tumor cultures. Our data showed that local treatment of tumor bearing animals with Ad5/3- E2F-d24-hIL7 significantly decreased cancer growth and increased frequency of tumor-infiltrating cells. Ad5/3-E2F-d24-hIL7 promoted notable upregulation of pro-inflammatory cytokines, and concomitant activation and migration of CD4+ and CD8 + T cells. Interleukin 7 expression within the tumor was positively correlated with increased number of cytotoxic CD4+ cells and IFNg-producing CD4+ and CD8+ cells. These findings offer an approach to overcome the current limitations of conventional IL7 therapy and could therefore be translated to the clinic.

Merkel cell carcinoma (MCC) is a rare cutaneous carcinoma that has gained enormous interest since the discovery of Merkel cell polyoma virus, which is a causative oncogenic agent in the majority of MCC tumours. Increased research has focused on effective treatment options with immuno-oncology. In this study, we reviewed the real-world data on different treatments given to MCC patients in Finland in 1986-2016. We used the Finnish Cancer Registry database to find MCC patients and the Hospital Discharge Register and the Cause-of-Death Register to obtain treatment data. We identified 376 MCC patients and 33 different treatment entities and/or combinations of treatment. An increase was noted in the incidence of MCC since 2005. Therefore, the cohort was divided into two groups: the "early" group with time of diagnosis between years 1986 and 2004 and the "late" group with time of diagnosis between 2005 and 2016. The multitude of different treatment combinations is a relatively new phenomenon; before the year 2005, only 11 treatments or treatment combinations were used for MCC patients. Our data show that combining radiation therapy with simple excision provided a survival advantage, which was, however, lost after adjustment for stage or age. Our registry study serves as a baseline treatment efficacy comparison as we move into the age of immunotherapy in MCC. Standardizing the treatment of MCC patients in Finland requires more work on awareness and multidisciplinary co-operation.